Cardiovascular disease is currently the leading cause of death in the United States. Approximately ninety percent of cardiovascular disease is presently diagnosed as atherosclerosis. Cardiovascular disease has been linked to several causative factors, which include hypercholesterolemia, hyperlipidemia, and the expression of VCAM-1 in vascular endothelial cells.
Hypercholesterolemia and Hyperlipidemia
Hypercholesterolemia is an important risk factor associated with cardiovascular disease. Serum lipoproteins are the carriers for lipids in the circulation. Lipoproteins are classified according to their density: chylomicrons, very low-density lipoproteins (VLDL), low density lipoproteins (LDL) and high-density lipoproteins (HDL). Chylomicrons primarily participate in transporting dietary triglycerides and cholesterol from the intestine to adipose tissue and liver. VLDL deliver endogenously synthesized triglycerides from liver to adipose and other tissues. LDL transports cholesterol to peripheral tissues and regulate endogenous cholesterol levels in those tissues. HDL transports cholesterol from peripheral tissues to the liver. Arterial wall cholesterol is derived almost exclusively from LDL. Brown and Goldstein, Ann. Rev. Biochem. 52, 223 (1983); Miller, Ann. Rev. Med. 31, 97 (1980)). In patients with low levels of LDL, the development of atherosclerosis is rare.
Elevated cholesterol levels are associated with a number of disease states, including restenosis, angina, cerebral atherosclerosis, and xanthoma. It is desirable to provide a method for reducing plasma cholesterol in patients with, or at risk of developing, restenosis, angina, cerebral arteriosclerosis, xanthoma, and other disease states associated with elevated cholesterol levels.
If it has been determined that hypercholesterolemia is due to elevated LDL (hyperlipidemia), the lowering of LDL levels by dietary therapy is attempted. There are several drug classes that are commonly used to lower LDL levels, including bile acid sequestrants, nicotinic acid (niacin), and 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors. Probucol and the fibrate derivatives are sometimes used as adjunctive therapy, usually in combination with other medications. The HMG CoA reductase inhibitors have been termed statins or vastatins. Statins are among the most effective agents currently on the market for hypercholesterolemia, and include pravastatin (Pravchol, Bristol Myers Squibb), atrovastatin (Warner Lambert/Pfizer), simvastatin (Zocor, Merck), lovastatin (Mevacor, Merck), and fluvastatin (Lescol).
For many patients, diet plus one of the hypolipidemic agents will be sufficient. However, for patients with an initial LDL cholesterol level of greater than 200 mg/dl, therapy needs to lower LDL levels by 50% or more. Although a single agent may occassionally achieve this degree of LDL lowering, it is far more common to see decreases of only 20 to 30%. Thus, for the patient with heterozygous familial hypercholesterolemia with an LDL cholesterol of 200 to 400 mg/dl, a combination of two, or occasionally, three hypolipidemic drugs will be required to achieve an LDL cholesterol level of less than 100 mg/ml. Combinations of a bile sequestrant resin and nicotinic acid can lower LDL levels by 45% to 55%, a resin plus a statin, by about 50% to 60%, nicotinic acid plus a statin by about 50%, and triple drug therapy, using a combination of a bile acid-binding resin, a statin, and nicotinic acid, by as much as 70%.
Evidence suggests that the atherogenic effects of low density lipoprotein (LDL) may be in part mediated through its oxidative modification. Probucol has been shown to possess potent antioxidant properties and to block oxidative modification of LDL. Consistent with these findings, probucol has been shown to actually slow the progression of atherosclerosis in LDL receptor-deficient rabbits as discussed in Carew et al. Proc. Natl. Acad. Sci. U.S.A. 84:7725-7729 (1987). Most likely, probucol is effective because it is highly lipid soluble and is transported by lipoproteins, thus protecting them against oxidative damage.
Probucol is chemically related to the widely used food additives 2,[3]-tert-butyl-4-hydroxyanisole (BHA) and 2,6-di-tert-butyl-4-methyl phenol (BHT). Its fill chemical name is 4,4'-(isopropylidenedithio) bis(2,6-di-tert-butylphenol).
Today, probucol is used primarily to lower serum cholesterol levels in hypercholesterolemic patients. Probucol is commonly administered in the form of tablets available under the trademark Lorelco.TM.. Unfortunately, probucol is almost insoluble in water and therefore cannot be injected intravenously. In fact, probucol is difficult for cells to absorb in vitro because of its poor miscibility in buffers and media for cell culture. Solid probucol is poorly absorbed into the blood, and is excreted in substantially unchanged form. Further, the tablet form of probucol is absorbed at significantly different rates and in different amounts by different patients. In one study (Heeg et al., Plasma Levels of Probucol in Man After Single and Repeated Oral Doses, La Nouvelle Presse Medicale, 9:2990-2994 (1980)), peak levels of probucol in sera were found to differ by as much as a factor of 20 from patient to patient. In another study, Kazuya et al. J. Lipid Res. 32; 197-204 (1991) observed an incorporation of less than about 1 .mu.g of probucol/10.sup.6 cells when endothelial cells are incubated for 24 h with 50 .mu.M probucol.
U.S. Pat. No. 5,262,439 to Parthasarathy discloses soluble analogs of probucol in which one or both of the hydroxyl groups are replaced with ester groups that impart water solubility to the compound. In one embodiment, the soluble derivative is selected from the group consisting of a mono- or di-succinic acid ester, glutaric acid ester, adipic acid ester, suberic acid ester, sebacic acid ester, azelaic acid, or maleic acid ester of probucol. In another embodiment, the probucol derivative is a mono- or di-ester in which the ester contains an alkyl or alkenyl group that contains a functionality selected from the group consisting of a carboxylic acid group, amine group, salt of an amine group, amide groups, amide groups, and aldehyde groups.
A series of French patents disclose that certain probucol derivatives are hypocholesterolemic and hypolipemic agents: Fr 2168137 (bis 4-hydroxyphenylthioalkane esters); Fr 2140771 (tetralinyl phenoxy alkanoic esters of probucol); Fr 2140769 (benzofuryloxyalkanoic acid derivatives of probucol); Fr 2134810 (bis-(3-alkyl-5-t-alkyl-4-thiazole-5-carboxy) phenylthio)alkanes; FR 2133024 (bis-(4-nicoinoyloxyphenythio)propanes; and Fr 2130975 (bis(4-(phenoxyalkanoyloxy)phenylthio)alkanes).
U.S. Pat. No. 5,155,250 discloses that 2,6-dialkyl-4-silylphenols are antiatheroscierotic agents. The same compounds are disclosed as serum cholesterol lowering agents in PCT Publication No. WO 95/15760, published on Jun. 15, 1995. U.S. Pat. No. 5,608,095 discloses that alkylated-4-silyl-phenols inhibit the peroxidation of LDL, lower plasma cholesterol, and inhibit the expression of VCAM-1, and thus are useful in the treatment of atherosclerosis.
Expression of VCAM-1
Adhesion of leukocytes to the endothelium represents a fundamental, early event in cardiovascular disease as well as in a wide variety of inflammatory conditions, including autoimmune disorders and bacterial and viral infections. Leukocyte recruitment to the endothelium is started when inducible adhesion molecule receptors on the surface of endothelial cells interact with counterreceptors on immune cells. Vascular endothelial cells determine which type of leukocytes (monocytes, lymphocytes, or neutrophils) are recruited, by selectively expressing specific adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin (ELAM). In the earliest stage of the atherosclerotic lesion, there is a localized endothelial expression of VCAM-1 and selective recruitment of mononuclear leukocytes that express the integrin counterreceptor VLA-4. Because of the selective expression of VLA-4 on monocytes and lymphocytes, but not neutrophils, VCAM-1 is important in mediating the selective adhesion of mononuclear leukocytes. VCAM-1 is involved as a mediator in chronic inflammatory disorders such as asthma, rheumatoid arthritis and autoimmune diabetes. For example, it is known that the expression of VCAM-1 and ICAM-1 are increased in asthmatics. Pilewski, J. M., et al. Am. J Respir. Cell Mol. Biol. 12, 1-3 (1995); Ohkawara, Y., et al., Am. J Respir. Cell Mol Biol. 12, 4-12 (1995). Additionally, blocking the integrin receptors for VCAM-1 and ICAM-1 (VLA-4 and LFA-1, respectfully) suppressed both early and late phase responses in an ovalbumin-sensitized rat model of allergic airway responses. Rabb, 11. A., et al., Am. J. Respir. Care Med. 149, 1186-1191 (1994). There is also increased expression of endothelial adhesion molecules, including VCAM-1, in the microvasculature of rheumatoid synovium. Koch, A. E. et al., Lab. Invest. 64, 313-322 (1991); Morales-Ducret, J. et al., Immunol. 149, 1421-1431 (1992). Neutralizing antibodies directed against VCAM-1 or its counter receptor, VI A-4, can delay the onset of diabetes in a mouse model (NOD mice) which spontaneously develop the disease. Yang, X. D. et al., Proc. Natl. Acad. Sci. U.S.A. 90, 10494-10498 (1993); Burkly, L. C. et al., Diabetes 43, 523-534 (1994); Baron, J. L. et al., J. Clin. Invest. 93, 1700-1708 (1994). Monoclonal antibodies to VCAM-1 can also have a beneficial effect in animal models of allograft rejection, suggesting that inhibitors of VCAM-1 expression may have utility in preventing transplant rejection. Oroez, C. G. et al., Immunol. Lett. 32, 7-12 (1992).
VCAM-1 is expressed by cells both as a membrane bound form and as a soluble form. The soluble form of VCAM-1 has been shown to induce chemotaxis of vascular endothelial cells in vitro and stimulate an angiogenic response in rat cornea. Koch, A. F. et al., Nature 376, 517-519 (1995). Inhibitors of the expression of soluble VCAM-1 have potential therapeutic value in treating diseases with a strong angiogenic component, including tumor growth and metastasis. Folkman, J., and Shing, Y., Biol. Chem. 10931-10934 (1992).
VCAM-1 is expressed in cultured human vascular endothelial cells after activation by lipopolysaccharide (LPS) and cytokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF-a). These factors are not selective for activation of cell adhesion molecule expression.
Subsequent conversion of leucocytes to foamy macrophages results in the synthesis of a wide variety of inflammatory cytokines, growth factors, and chemoattractants that help propagate the leukocyte and platelet recruitment, smooth muscle cell proliferation, endothelial cell activation, and extracellular matrix synthesis characteristic of maturing atherosclerotic plaque.
Molecular analysis of the regulatory elements on the human VCAM-1 gene that control its expression suggests an important role for nuclear factor-kB (NF-kB), a transcriptional regulatory factor, or an NF-kB like binding protein in oxidation-reduction-sensitive regulation of VCAM-1 gene expression. Transcriptional factors are proteins that activate (or repress) gene expression within the cell nucleus by binding to specific DNA sequences called "enhancer elements" that are generally near the region of the gene, called the "promoter," from which RNA synthesis is initiated.
The promoters for both VCAM-1 and ICAM-1 have been cloned and characterized. For example, both promoters contain multiple DNA sequence elements which can bind the transcription factor, NF-kB. Iademarco, M. F. et al., J Biol. Chem. 267, 16323-16329 (1992).
Nuclear factor-kB is a ubiquitously expressed multisubunit transcription factor activated in several cell types by a large and diverse group of inflammatory agents such as TNFa, IL-1B, bacterial endotoxin, and RNA viruses. It plays a key role in mediating inflammatory and other stress signals to the nuclear regulatory apparatus. Although the precise biochemical signals that activate NF-kB are unknown, this transcriptional factor may integrate into a common molecular pathway many of the risk factors and "causative" signals of atherosclerosis, such as hyperlipidemia, smoking, hypertension, and diabetes mellitus.
The activation of NF-kB in vascular endothelial cells by diverse signals can be specifically inhibited by antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate. This has led to the hypothesis that oxygen radicals play an important role in the activation of NF-kB through an undefined oxidation-reduction mechanism. Because an NF-kB-like enhancer element also regulates the transcription of the VCAM-1 promoter in an oxidation-reduction-sensitive manner, it was hypothesized that oxidative stress in the atherosclerotic lesion may play a role in regulating VCAM-1 gene expression through this oxidation-reduction-sensitive transcriptional regulatory protein. U.S. Pat. No. 5,380,747 (PCT/US93/10496) disclosed for the first time that the expression of VCAM-1 in vascular endothelial cells can be inhibited by the administration of a class of dithiocarbamates, which include pyrrolidine dithiocarbamate. These dithiocarbamates are thus useful in the treatment of cardiovascular disease, and have now been shown to significantly reduce the presence of atherosclerotic lesions in hypercholesterolemic rabbits.
It has been hypothesized that modification of low-density lipoprotein (LDL) into oxidatively modified LDL (ox-LDL) by reactive oxygen species is the central event that initiates and propagates atherosclerosis. Steinberg, et al., N. Engl. J. Med. 1989; 320:915-924. Oxidized LDL is a complex structure consisting of at least several chemically distinct oxidized materials, each of which, alone or in combination, may modulate cytokine-activated adhesion molecule gene expression. Fatty acid hydroperoxides such as linoleyl hydroperoxide (13-HPODE) are produced from free fatty acids by lipoxygenases and are an important component of oxidized LDL.
It has been proposed that a generation of oxidized lipids is formed by the action of the cell lipoxygenase system and that the oxidized lipids are subsequently transferred to LDL. There is thereafter a propagation reaction within the LDL in the medium catalyzed by transition metals and/or sulfhydryl compounds. Previous investigations have demonstrated that fatty acid modification of cultured endothelial cells can alter their susceptibility to oxidant injury. PCT/US95/05880 disclosed that polyunsaturated fatty acids and their hydroperoxides induce the expression of VCAM-1, but not ICAM-1 or E-selectin in human aortic endothelial cells, through a mechanism that is not mediated by cytokines or other noncytokine signals. This was a fundamental discovery of an important and previously unknown biological pathway in VCAM-1 mediated immune responses. It was also reported in PCT/US95/05880 that the induction of VCAM-1 by polyunsaturated fatty acids and their hydroperoxides is supressed by dithiocarbamates, including pyrrolidine dithiocarbamate.
Given that cardiovascular disease is currently the leading cause of death in the United States, there is a need to provide new therapies for its treatment. It is a goal to provide new agents that can simultaneously treat hypercholesterolemia, hyperlipidemia, and can inhibit the expression of VCAM-1 in vascular endothelial cells.
Therefore, it is an object of the present invention to provide a method and composition for suppression of VCAM-1, and in particular a method for the treatment of cardiovascular disease.
It is a further object of the present invention to provide a method and composition for the treatment of cardiovascular disease that can simultaneously treat hypercholesterolemia, hyperlipidemia, and can inhibit the expression of VCAM-1 in vascular endothelial cells.